DE2009342B2 - Use of glycerol alkyl ether (l) phosphoric acid (3) monocholine esters - Google Patents

Description

CH ₁ -O-PO ₁ -CH ₁ -CH ₁ -N (CH,)., (D

I)

in which R is an alkyl radical with at least 12 Designated carbon atoms, in increasing the natural resistance of the organism.

2. Use of a glycerol-alkyl ether- (l) -phosphoric acid- (3) -monocholine ester of the general- _> » nice shapes! ', where R is an alkyl radical with IS Designated carbon atoms, according to claim 1.

3. Glycerol-octadecylätherf, I) -phosphoric acid (3) -monocholine ester.

The invention relates to the use of glycerol alkyl ether-ilJ-phosphoric acid-pI-monochoIin- κ ι esters (ether-lysolecithins) of the general formula

CH ₂ O-R

CHOH, -,

CU ₁ O PO ₂ CH ₂ CH ₂ N (CH,)., (I)

O ₁ -,

in

in which R denotes an alkyl radical having at least 12, preferably 18, carbon atoms, on the increase the natural resistance of the organism.

So far, a large number of studies have been carried out on the living cell, the smallest unit of the organism. One knows very well the morphological structure of the cell and essentially knows about the meaning and function of the individual subcellular structures. The cell membrane has also already been the subject of detailed investigations. One has certain ideas about their fine structure and numerous membrane functions, such as active or passive transport, hindered or facilitated diffusion, fluxes, pulse effects, excitation processes, signal processing, and the like have already been thoroughly studied. It is all the more surprising that so little attention has hitherto been paid to the interface properties of the membranes, although it is easy to see that many, if not the predominant part, of the membrane functions are decisively influenced by the interface activity of the membranes. The idea that the central control and regulation mechanism is to be seen in the change in the interface activity is not so absurd if one keeps in mind that changes in the interface activity not only affect the permeability of the membranes, but also those that occur there electrical potential thresholds are influenced. If one also takes into account that the permeability of the membrane is responsible for the direction, amount and type of the substances to be transported by the wind and that the membrane potential plays an important role in the transmission of signals in the organism - it should not be forgotten that changes in potential differences in membranes are ultimately based on their changes in permeability - so one could draw the conclusion that it ought to be possible to produce targeted biological effects by influencing the activities of the membrane interfaces.

However, it would be regarded as absurd in the specialist field to influence the interfacial activities with the aid of surface-active substances want, because it is known that surface-active or interface-active substances are generally highly toxic substances. Degrade substances of this type already in concentrations that are far below the dose range that is sufficient to achieve a biological or pharmacodynamic effect, the interfacial activity of the membrane so far that its disintegration occurs. This means that surface-active substances generally have a cytolytic effect, they destroy the membrane and thereby destroy the cell. The cytolytic effect is all the more so be larger, the more the surface-active substances are absorbed, d. H. attached to the membrane, and ever less and the slower they are metabolized.

Lysolecithins, which are acylglycerol phosphoric acid choline esters, also have surface-active substances Properties and it is known that in higher concentrations they are cytolytic, e.g. B. hemolytic, act. Nevertheless, the lethal dose of lysolecithins is relatively high, which is due to the fact that lysolecithins extremely rapidly in the organism, among other things. by Reacylation too. ecithincn or by deacylation be metabolized to glycerylphosphorylcholine. It is all the more astonishing that the ether-lysolecithinc Formula I, which are also surface-active and which differ from the lysolecilhines in that the acyl group is replaced by an alkyl group and therefore should not be metabolizable or at least significantly more difficult to metabolize, are also relatively non-toxic. they show dose-dependent pharmacodynamic effects already in a dose range which is considerably below the Value of the dose lctalis lies. As surface-active substances, the compounds of the formula I also change the Membrane surface activity and they are therefore excellent in influencing and Renewal of the surface area of membranes.

From the publication "Modern Medicines" by I. B. Hellwig. Stuttgart, 1967, is already a number of compounds known to which a cylological or hemolytic effect is said and which could therefore be viewed as unspecific irritants. But there is no mention of these connections became known that they can be used, for example, in the treatment of Tiimor, im On the contrary, with some of these compounds tumors are considered contraindications.

There are already in 1.iebigs Ann. Chcm., 709 (1967), 2J4-239, compounds have been described which are within the range of the formula given here. Above however, nothing is said about their effectiveness in increasing natural resistance.

Ks now became tfcfuruk-ii. that ether l.vsolccithinc

of formula I in increasing the natural Let resistance of the organism use. This increase in resistance shows in an increased form Resistance to infections as well as to tumors, furthermore in the potentiation of the Antigenicity of weak, otherwise tolerated immunogens. In addition, the compounds of the formula I can also be used as auxiliaries for attracting cells in vivo and used to obtain highly active immunocompetent cells.

The representation of the compounds of formula I is known from the literature and can, for. B. after that of D. Arnold, H. U. Weltzien and O. Westphal in Liebigs Ann. Chem, 709, 234 (1967). the The synthesis proceeds according to the reaction scheme below, starting from 1,3-benzylidene glycerol Via glycerine benzyl ether (2), alkylation, reaction of the glycerine diet with phosphoric acid mono-2-bromoethyl ester dichloride and introduction of the trimethylamine residue Glycerine alkyl ether (l) benzyl ether (2) phosphoric acid choline ester, from which the benzyl protective group is then removed by hydrogenolysis, as will be explained in more detail below.

CHOH CHC ₆ H,
CH, - O

CH ₁

«(
( H
Hi H Il S. Il O Bz CH CH,
/
O CU ₂ CU ₂ cn. i
I! "II. OH > CU
I.
CU ₁ cn. O
OH B / CII ₂ • < O Alkyl • ( O Ii /. OH O Alkyl O Ii /. O PO ₂ CIl ₂ CII, Ur Cl O
O Alkyl
H/ O PO ₂ CII ₂ CII, Mr.
on

CH ₂ -O-alkyl
- »CH-Ο —Bz

l

l + l

CH-O-PO ₂ -CH ₃ -CH ₂ -N (CH,).,

Alkyl = alkyl radical with at least 12 carbon atoms Bz = CH ₂ -QsH ₅ , benzyl group.

The compounds of formula I have an asymmetric carbon atom, they can therefore in two stereoisomeric forms occur. The preparation of the stereoisomeric compounds can be carried out by appropriate Modification of the above reaction scheme, d. H. Using optically active compounds, or through subsequent cleavage of the final reaction product into the optical antipodes take place.

The preparation of the compounds is based on octadecyl-lysolecithin, which has not yet been described in the literature explained

Manufacturing example
j ',

Glycerol octadecyl ether f 1) phosphoric acid (3) monocholine ester

a) Glycerin-octadecylether- (l) -benzylether- (2)

κι 14 g (0.07 mol) of CAycerol benzyl ether (2) are in 500 ml of absolute xylene dissolved, 1.62 g (0.07 mol) of sodium were added and the mixture was gradually increased to 100.degree heated. After 6 hours, after a thick suspension of sodium alcoholate has formed. 34 g (0.09

r. Mol) n-octadecyl iodide added and another 6 Stirred at 90 ° C. for hours. The solution becomes clear at first, but gradually begins after a while Sodium iodide to precipitate. After cooling and adding 500 ml of water, the organic phase is poured

in separated. All components are distilled off from it, which are volatile at 0.1 torr up to 180 ° C. It 29.2 g of crude product remain. These are in three portions on 120 g each of silica gel with ether / petroleum ether (1: I) as mobile phase chromatography. Yield to

• η pure diether: 8.2 g (corresponding to 19% of theory) as a colorless, waxy substance with melting point 45 ° C. (R. = 037).

Analysis for C _2n HwO _s (434.7).

Ber. C 77.36 H 11.59
Found C 77.03 H 11.27

b) Glycerol-octadecylether-flJ -benzylether - ^) - phosphoric acid- (3) -monocholine ester

6.8 g (0.016 mol) of glycerol octadecyl ether OVbenzyl ether (2) are dissolved in 60 ml of absolute chloroform and the solution at room temperature with 4.0 g (0.017 mol) of phosphoric acid mono (2) bromoethyl ester dichloride and 3.8 g (0.065 mol) of triethylamine in 60 ml of chloroform were slowly added dropwise. After standing for one day at room temperature, the red-brown solution is vigorously stirred for hydrolysis with 8 ml of water for 2 hours. After evaporation in vacuo the residue in a high vacuum over phosphorus pentoxide is extracted dried and then with 300 ml of absolute petroleum ether (60 -70 ⁰ C). The ammonium salts that have remained undissolved are filtered off with suction and evaporated

Flitrat, dissolve the residue in 100 ml of chloroform and Shake vigorously for 5 minutes with 20 ml of saturated aqueous barium acetate solution. The cleaning of the The barium salt is carried out on 200 g of silica gel with chloroform / methanol (9: 2) as the mobile phase, the main product being is obtained as a colorless gummy substance (Ri = 0.58). After falling over with acetone from ether (both absolute) one receives 6.2 g of pure barium salt. This is now in 150 ml of absolute methanol (about 50 - 60 ° warm) Shaken for 15 minutes with 10 ml of air-dried cation exchange resin in the Η shape, gradually taking Solution occurs. After suctioning off and stripping off the solvent, 5.8 g of free bromoethyl phosphate are obtained as a colorless oil. This is then boiled with 300 ml of a 12.5% methanolic trimethylamine solution 6 hours at reflux. The solid lecithin bromide (7.0 g) obtained after evaporation is then vigorously stirred in 200 ml of methanol with 2.8 g of silver acetate for 30 minutes at room temperature. Included creates a waxy raw lecithin, from which the last traces of silver acetate can only be found by chromatography on 200 g of silica gel with chloroform / methanol / water (65: 25: 4) as the mobile phase can (Rf = OpB) - After falling over with acetone from little In chloroform, the pure product is finally obtained as a colorless powder in a yield of 40% of theory (over both levels).

Analysis for Cj ₃ HmO ₇ NP (617.9).

Ben C 64.15 H 10.44 N 2.27 P 5.01
Found C 63.40 H 10.26 N 2.32 P 5.07

c) Glycerol octadecyl ether (1) phosphoric acid (3) monocholine ester

1.0 g (1.62 mmol) of glycerol-ocstadecyläther-OJ-benzyläther- (2) -phosphorsäure- (3) -monocholinester are mixed with 250 mg of palladium black in 150 ml of absolute methanol for 7 hours at 40 ⁰ C and under atmospheric pressure hydrogenated. Subsequent chromatography on 100 g of silica gel with chloroform / methanol / water in a volume ratio of 65: 25: 4 yields the pure ether lysolecithin as a colorless powder with an Rr value of 0.2. The yield is 0.64 g, corresponding to 75% of theory.

Analysis for C ₂₆ H ₅₈ O ₇ NP (527.7).

Ber. C 59.17 H 11.08 N 2.65 P 5.87
Found C 57.69 H 11.26 N 2.48 P 5.60

Comparative examples to the natural
Increase in resistance

1. The increase in resilience
against infections

Was determined by determining the survival time of mice after infection with pathogens in Based on that described by H. G. Howard, D. R. Rowley and A. C. Wardlaw in Nature, 179 (1957), 317 Test arrangement checked. Animals given before intraperitoneal infection with E. coli 145 ether lysolecithins of formula I i. p. had a survival line two to three times longer than that untreated control animals. The reason for the increase in resistance can be an increased phagocytosis be seen. Animals pretreated with ether lysolecithin showed increased uptake of germs in d; .J cytoplasm of phagocytes (macro- and microphages). Both the number of phagocytic cells and the number consumed per cell in 3 bacteria were increased several times. These findings could be made both in vivo and in vitro

■ s are collected.

The example of fmpftun.oren could increase the resistance to tumors be detected. The so-called mouse Ehrlich ascites tumor is an increasing one in all mouse strains

m intraperitoneal vaccine tumor that appears after about 10 to 14 Days leads to the death of all vaccinated animals, but if the animals are treated with ether lysolecithins of the formula I pretreated, so the growth of the tumor could be completely prevented in 50% of the test animals,

ι · 5 d. H. the transferred tumor was destroyed in the In the remaining 50% of the animals, a two to three fold increase in survival was observed.

2. The potentiation of the antigenicity of weak immunogens,

which likewise as a disturbance of the natural Resistance to be considered kurtn was tested as follows:

a) The test was based on the method of Dresser (Immunology, 9 [1965], 261). The basis ■ the experimental arrangement consists in the induction of Soluble protein tolerance. In this experimental set-up, the ability of substances investigates the immune response in the organism to the extremely weak immunogenic bovine Gamma globulin (BGG) to be amplified so that antibodies against this protein are properly detected can be. Mice receive an intraperitoneal injection of centrifuged aggregate-free BGG at a dose of 5 mg. Usually at this dose there will be no antibodies after 8 to 10 days verifiable. So the animals are not immunized. Under these conditions you are incapable of opposing there ;; BGG to give an immune response. If, on the other hand, the BGG is given in combination with an adjuvant. so will the temporary tolerance development is prevented and the animals now form antibodies against the otherwise tolerogenic BGG. 10 to 12 days after administration of the tolerogenic protein, the animals are injected again with BGG, the is labeled with iodine-125. If the animals are tolerant, so will the labeled antigen slowly degraded like its own gamma globulin

If the animals are immune to this, what is known as immune elimination occurs. i.e., the labeled antigen will removed from the cycle much faster. The measure for the antibodies formed is therefore used Rate of elimination of iodine-125 labeled BGG.

When testing the octadecyl ether lysolecithin, it was found. that opposite one the animals treated with BGG and ether lysolecithin only with the control group pre-injected with sodium chloride eliminate the tracer protein from the circulation ten to one hundred times faster.

b) Another immunological determination method for antibodies, with the help of which potentiation the antigenicity of weak immunogfne can be determined, is based on the fact that the antigen (BGG) Erythrocytes are coupled and the cells treated in this way with the serum in a geometric manner The dilution series can be incubated for 20 hours at 4 ° C. If the serum contains antibodies, so the erythrocytes are aEElutinated. The highest

Serum dilution at which this phenomenon can still be observed is called an antibody.

The use according to the invention could also be confirmed with this, considerably more imprecise method.

The compounds of the formula I can be applied in a customary manner. The is particularly suitable intraperitoneal injection. The dose can vary within wide limits and can be from about 0.5 to about 10 mg / kg.

3. Comparative studies on the effectiveness of l-DL-octadecyl-2-hydroxy-phosphorylcholine in myeloma

It became the anti-tumor efficacy of

1. Bacteria Calmette Guerin (BCG)
comparing with

2. l-DL-octadecyl ^ -hydroxy-phosphorylcholine

and untreated animals in myeloma X5563 in C3H mice tested.

10 animals each received 7 days before the tumor implantation

tion intraperitoneally either t χ 10 'live bacteria Calmette Guerin injected or the ether-lysolecithin on the 1st or 5th day after tumor implantation in a Dosage of 50 μg per animal orally. This procedure is common in tumor tests with mycobacteria on the one hand and lysolecithins on the other.

34 days after the inoculation of the myeloma, the following final result was recorded:

It survived in the groups

untreated control I animal Animals with BCG 4 animals Animals with ether-isolccithin on day I. 8 animals Animals with ether-Iysolecithin on Ί ag 5 7 animals

In the animals pretreated with BCG, im Compare / u the lysolccithin groups the tumors 10 Days earlier and grew twice as fast.

The test result is clear and reproducible.

Claims (1)

Patent claims: 1. Use of glycerol-alkylether- (l) -phosphoric acid- (3) -monochoIinester of the general formula CFi ₂ -OR CHOH